Chlamydia trachomatis, composed of 15 odd serovars (A through K and L1 to L3) with serovars D through K and the lymphogranuloma venereum strains L1 - L3 causing sexually transmitted diseases (STDs), is the most common bacterial cause of STDs and if untreated often lead to severe complications, including pelvic inflammatory disease, ectopic pregnancy and infertility in women. Since most infected individuals do not seek treatment due to lack of obvious symptoms, prophylactic immunization will be the best option for preventing infection and controlling chlamydial diseases. An efficacious chlamydial vaccine should induce broad-based, long lasting immunity against the different chlamydial serovars. We previously showed that the recombinant Vibrio choerae ghost (rVCG) platform is an effective delivery system capable of simultaneously delivering multiple Chlamydia antigens to the immune system. The results suggested that achievement of a long-term, cross-protective chlamydial immunity is possible and would require the mucosal delivery of specific multiple chlamydial antigens and effective immunomodulation of host immune response. Thus, the goal of this proposal is to utilize an rVCG- based multisubunit vaccine candidate together with a molecular mucosal adjuvant to validate a mucosal route for vaccine delivery and enhancement of chlamydial immunity. We will examine how a combination of the mucosal route of administration and mucosal adjuvant affect the profile and quality of antibody and T cell response for protection against Chlamydia. We propose to test the overall hypothesis that intrarectal (IR) delivery of select multiple chlamydial antigens together with a molecular adjuvant will enhance genital tract immunity and provide broad-based, long-lasting protection against intravaginal challenge. In specific aim 1, we will establish that IR delivery of an rVCG-based vaccine together with Flt3 ligand (FL) will enhance long-term, cross- protective chlamydial immunity. Specific aim 2 will examine the effect of FL adjuvant on the profile and quality of immune effectors elicited by a chlamydial vaccine after rectal mucosal delivery. Finally, studies in Specific aim 3 will elucidate the molecular mechanisms involved in the induction of immune responses by an rVCG vaccine expressing FL and determine the migration pathway of antigen presenting cells after IR delivery of rVCG vaccine. PUBLIC HEALTH RELEVANCE: Genital Chlamydia infection is the most common bacterial sexually transmitted disease (STD) worldwide. Women usually have few or no symptoms associated with infection, which often leads to delays in treatment and the development of infectious complications such as pelvic inflammatory disease and infertility and increased likelihood of acquiring HIV infection. These studies will provide a better understanding of the antigenic and immunologic correlates of broad-based, long-lasting protective immunity against Chlamydia and will guide the rational development of vaccine strategies that will aid in reducing the economic burden on the public healthcare system due to high treatment costs of infectious complications.